The non-chromosomal genes URE3, PSI and PIN of Saccharomyces cerevisiae are infectious forms (prions) of the Ure2p, Sup35p and Rnq1p, respectively, while Het-s of Podospora anserina is a prion of the HETs protein (reviewed in 1). The basis of each of these prions is a self-propagating amyloid of the respective protein, as the mammalian infectious protein PrPSc is believed to be an amyloid of the cellular PrP protein. Amyloid is not suitable material for either X-ray crystallography or solution NMR structural studies but can be studied by solid-state NMR (2). We have undertaken solid-state NMR studies of each of the yeast and fungal prion amyloids in collaboration with Dr. Robert Tycko of LCP, NIDDK. We find that each of the yeast infectious prion amyloids, Sup35p, Ure2p and Rnq1p, the basis of the PSI, URE3 and PIN prions, respectively, are parallel in-register beta-sheet structures (3, 5, 6). Sup35p is a subunit of the translation termination factor, consisting of an N-terminal prion domain (N, residues 1-123), a middle highly charged domain (M, residues 124 - 253) and a C-terminal domain (C) that is sufficient for translation termination. We prepared infectious recombinant Sup35NM labeled with Tyr-1-13C, or Leu-1-13C, or Phe-1-13C, and made beta sheet-rich amyloid filaments from each. Beta sheets may be antiparallel, parallel or beta helices. Parallel beta sheets may be either in-register or out of register. An in-register beta sheet has identical residues aligned along the filament. For example, Val31 of the first molecule is 4.7 angstroms (the distance between beta strands in a beta sheet) from Val31 of the second molecule and so on down the fiber. We used a constant-time finite-pulse radio frequency driven recoupling method (4) to measure the distance from each labeled carbonyl carbon to the next nearest labeled nucleus. We found that for Tyr-1-13C and for Leu-1-13C, the decay indicated a distance of about 5 angstroms, demonstrating a parallel beta sheet structure, either in register or out of register by at most one residue (3). Diluting the fully labeled molecules with four parts of unlabeled molecules resulted in substantial decrease in the rate of signal decay, indicating the the interaction being observed was intermolecular, not intramolecular, and again supporting the parallel in-register structure. Ether-precipitated Sup35NM labeled with Leu-1-13C showed a slow decay rate indicating that the results observed were due to the specific amyloid structure, not simply due to aggregation. To confirm that the parallel sheet was indeed in-register, we made Sup35NM labeled with alanine-3-13C, that is, methyl labeled instead of carbonyl carbon labeled as in the other experiments. Because amino acid R groups point in alternate directions with succeeding residues on a beta strand, a parallel beta sheet out of register by a single residue would give a very slow decay rate. In fact the results indicated an in-register structure (3). This was the first structure determined for any prion amyloid. Further work will be needed to determine the details of this structure, but specifying a parallel in-register structure constrains the possibilities very tightly. We have now carried out similar studies on infectious amyloid of the Ure2 prion domain (residues 1-89) with similar results (5). Similarly, we have shown that infectious amyloid of the Rnq1 protein, the basis of the PIN prion of yeast has a parallel in-register beta-sheet structure. Earlier work had shown, surprisingly, that shuffling the prion domain of Ure2p or of Sup35p did not prevent the corresponding proteins from becoming prions (7, 8). We inferred from this fact that the structures must be parallel in-register beta sheets. As mentioned above, this has now been verified for the normal Ure2p and Sup35p prion domains, and we have also shown the same for the shuffled prion domains (9). This confirms that for these prions, it is amino acid composition, not sequence, that determines ability to be a prion. The same amino acid sequence can form biologically distinct, heritable prion variants, which must represent structures differing in some way. We have examined two prion variants of Sup35p, and find that both are parallel in-register beta sheet structures (10). We propose that the differences between variants are in the locations of folds of the beta sheets and in the extent of the beta sheet. Implications: In the parallel in-register beta sheet structure, each residue of the prion domain contacts the same residue in the molecule that was laid down before it and the same residue in the next molecule to join the filament. This provides the possibility of passing on structural variations - such as turns of the sheet or irregularities in the structure - from parent molecules (already in the filament) to daughter molecules newly being laid down. This is the essential "templating" mechanism that must be present for these proteins to act as genes. This can explain the prion "variants", cases where the identical amino acid sequence produces different phenotypes or prion stabilities, doubtless due to differing details of the amyloid structure. This structure also indicates that the sequence of events is not, as often suggested, a primary conformational change of the prion protein, followed by aggregation. It seems evident that the conformational change is coincident with and a consequence of the new molecule joining the filament, and the details of the conformational change are directed by the structure of the end of the filament. 1. Wickner, R. B., H. K. Edskes, et al. (2007). "Prions of fungi: inherited structures and biological roles." Nat. Microbiol. Rev. 5: 611-618. 2. Tycko, R. (2006). Molecular structure of amyloid fibrils: insights from solid-state NMR. Q. Rev. Biophys. 1, 1-55. 3. Shewmaker, F., Wickner, R.B., and Tycko, R. (2006). Amyloid of the -sheet structure.&#61538;prion domain of Sup35p has an in-register parallel Proc. Natl. Acad. Sci. U. S. A. 103, 19754 - 19759. 4. Balbach, J.J., Petkova, A.T., Oyler, N.A., Antzutkin, O.N., Gordon, D.J., Meredith, S.C., and Tycko, R. (2002). Supramolecular structure in full-length Alzheimer's beta-amyloid fibrils: Evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance. Biophys. J. 83, 1205-1216. 5. Baxa, U., R. B. Wickner, A.C. Steven, D. Anderson, D., L. Marekov, W.-M. Yau, R. Tycko, R. (2007) Characterization of beta-sheet structure in Ure2p1-89 yeast prion fibrils by solid state nuclear magnetic resonance. Biochemistry 46: 13149 - 13162. 6. Wickner, R. B., F. Dyda, et al. (2008). "Amyloid of Rnq1p, the basis of the PIN+ prion, has a parallel in-register beta-sheet structure." Proc Natl Acad Sci U S A 105: 2403 - 2408. 7. Ross, E. D. et al. (2004). "Scrambled prion domains form prions and amyloid." Mol Cell Biol 24: 7206-7213. 8. Ross, E. D. et al. (2005). "Primary sequence independence for prion formation." Proc Natl Acad Sci U S A 102: 12825-12830. 9. Shewmaker, F., Ross, E. D., Tycko, R. and Wickner, R. B. (2008) Amyloids of shuffled prion domains that form prions have a parallel beta-sheet structure. Biochemistry 47:4000-4007. 10. Shewmaker F, Kryndushkin D, Chen B, Tycko R &Wickner RB (2009) Two prion variants of Sup35p have in-register beta-sheet structures, independent of hydration. Biochemistry 48: 5074-5082.